mica antibodies in renal transplantation

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The role of MIC-A in renal allograft loss - More than meets the eye?

Christos Argyropoulos

Renal Grand Rounds 12/1/08


The Case of Mr RC I

Patient is a 45 y/o wm diagnosed with LCD in April 2000.

a) Bone marrow biopsy (2000): increase in the number of bone marrow plasma cells (7%), with normal cytogenetics.

b) Native kidney biopsy : the glomeruli were hyperlobular and had eosinophilic material ( Congo red and Thio-T stains were negative) and wrinkling of the basement membrane . There was marked mesangial cellularity, with mesangial and tubular material deposition. Immunofluorescence staining of glomerular and tubular membranes was positive for kappa light chains and C3 was negative (C4 was not done).

At that time he received 4 doses of VAD chemotherapy which led to AKI from which the patient never recovered and was started on Hemodialysis in 8/00.

Patient transplanted in 4/10/6 and was maintained on FK/MMF since then.

Graft characteristics (DD, age 43, CIT1641, mismatched for A1,B1,DR2)

Patient’s baseline Scr between 1.5 – 2.1 up to 8/07


The Case of Mr RC II

From 9/07 until 8/05/08 patient’s Scr increased gradually up to 3.3

Rapid deterioration in renal function noted at the beginning of August: 3.8(8/7/08)→5.1 (8/11/08).

ELISA Class I,II screens : 0/0

Luminex Class I,II : (-)/weakly (+)

Luminex MICA : weakly (+)

Cylex: 371

Urine Protein/Creatinine ratio : 66/87.7


The case of Mr RC III

Allograft Biopsies (x2) :

GLOMERULOPATHY WITH INCREASED MESANGIAL CELLULARITY AND INCREASED MESANGIAL MATRIX

NO EVIDENCE OF ACUTE REJECTION (i0 t0 v0 g0).

MILD CHRONIC ALLOGRAFT NEPHROPATHY (ci1 ct1 cv1).

MILD ISOMETRIC TUBULAR VACUOLIZATION WITH SCATTERED PROTEIN RESORPTION DROPLETS

C4D stain was negative.

Borderline ACR noted in the first biopsy

On the basis of this biopsy and the positive MICA screen patient was treated as a possible MICA releated rejection with SM pulses.

Patient is pulsed with steroids on 8/0808 with reduction in serum creatinine to 3.5 (8/15/08).


Discovered in 1994 by a GenBANK search for sequences related to the MHC class I genes.

Named MIC (MHC class I chain related genes) due to their chromosomal location (6p21.3 in a 200kb region spanning the TNFA/B cluster, close to the HLA-B locus)

MICA encodes a 383-aa cell membrane polypeptide with a molecular weight of ~ 43 kDa.

MICA is highly polymorphic in its extracellular domain and at strong linkage disequilibrium with HLA haplotypes in specific populations.

MICA does not interact with the T Cell Receptor (TCR) but with NKG2D (C-type lectin activatory receptor) found in NK cells, Tγδ and CD8+ Tαβ cells.

MAJOR HISTOCOMPATIBILITY COMPLEX CLASS I CHAIN-RELATED GENE A: MICA


Transcription Pattern of MIC-A in Normal Human Tissues and Tumours

PLoS ONE. 2007; 2(6): e518. doi: 10.1371/journal.pone.0000518.


Effector functions of NKG2D engagement by NKG2DL (MICA)

Engagement of NKG2D by MICA leads to enhanced cytolytic activity of NK and Tγδ cells

This activity is directed against virally infected (e.g CMV) and tumour cells in multiple experimental systems.

Exposure to carcinogens upregulates MICA expression, while NKG2D-deficient mice develop tumours

Cell mediated killing through NKG2D is inhibited in the presence of HLA-I molecules (“missing-self”).

Engagement of NKG2D is transduced by DAP10 (a S-S bonded homodimer) that features a YXXM motif that interacts with P85 PI3K.In CD8(+) T cells, NKG2D may plays the same role as the the co-stimulatory molecule CD28.NKG2D (but not CD28) engagement induces Immunological Synapse formation in T cells.NKG2D signaling is enhanced by IL-15 or high doses of IL-2 converting T cells into NK like cytotoxic effector cells


Signal Transduction cascade of NKG2D signaling in NK cells

Trends in Molecular Medicine Vol.14 No.4 179-189


NKG2D ligands and their regulation

Trends in Molecular Medicine Vol.14 No.4 179-189


Non-HLA Antibodies In Renal Transplantation

Mismatch against non-HLA antigens can lead to rapid graft loss in experimental models of skin, and cardiac transplantation.

In clinical renal transplantation >10% of allograft biopsies with C4d deposition will not be associated with anti-HLA antigens.

It is estimated that 38% of chronic graft loss is due to antibody formation against non-HLA targets.

Candidate clinically relevant non-HLA antigens are present in non-lymphocyte cellular populations (e.g. the Endothelial – Monocyte (EM) antigen system.

Routine cross-matches are performed against T-lymphocytes not endothelial cells, hence non-HLA mismatch will go unrecognized.


Anti-MICA as non-HLA Abs

MICA molecules are expressed in endothelial cells which is the target of the recipient's immune response in acute (and chronic) humoral rejection

The MICA system is highly polymorphic and thus there is a high probability that a donor and the recipient will be mismatched for these antigens.

If the MICA system is implicated in graft loss, the following conditions should be met:

a. Antibody formation should precede organ dysfunction

b. Antibody formation should predict organ dysfunction

c. Antibody and/or antigen/antibody complex should be eluted from the target organ during episodes of acute dysfunction

d. There should be an approximate dose - response curve between antibody level and progression of organ dysfunction


Immunologically mediated tissue injury vs rejection

Conditions 1-4 are necessary conditions for an immunologic mechanism of tissue tissue injury.

These conditions are met by clinically important antibodies in acute and chronic antibody mediated diseases : ITP, AIHA, serum sickness, SLE, anti-GBM disease, post-infectious GN, Reiter's syndrome, Heymann nephritis, Goodpasture syndrome, immune hydrops fetalis, hyper-acute allograft rejection.

Autologous (anti-GBM disease), heterologous (post-infectious GN) or allogenous (anti-HLA) antibodies may lead to immunologically mediated tissue injury.

For an antibody to qualify as responsible for allograft rejection, its specificity against the donor (Donor Specific Antibody - DSA), rather than the recipient should be established.


Consequences of non HLA AB binding to the graft

Current Opinion in Immunology 2008, 20:607–613

a. Antibody formation

b. EC activation

c. Expression of CAM

d. Expression of cytokines

e. Activation of coagulation cascade

f. Migration of inflammatory cells to the graft

g. Tissue destruction via complement activation, thrombosis, cytolysis (relevant to MICA)


First clinical Studies of MICA Ab testing in renal transplantation I

First study (1) that examined the positivity of MICA in allograft recipients published in 2000

(1) Human Immunology 61, 917–924 (2000)

In 3/56 renal transplant recipients the specificity of the MICA antibody was determined : they harbored a MICA alloantibody i.e. a DSA.

Caveat : cross-reactivity and specificity of MICA-Abs not rigorously assessed. In 2/3 patients the investigators did not test the patient's own MICA for cross - reactivity


First clinical Studies of MICA Ab testing in renal transplantation II

In one patient multiple serum specimens were available allowing a longitudinal assessment.


Rejection


First clinical Studies of MICA Ab testing in renal transplantation III

Retrospective analysis of 748 serum samples from 139 pts (115 first grafts) assessed at various points after transplant.

69 (49.6%) of patients had one or more ACR (within the first 3 mos)

Detection of MICA antibodies: flow, blocking, WB

Ability of MICA-Ab to induce a prothrombotic phenotype on renal endothelial cells was assessed

(1) Transplantation Vol. 74, 268–277, No. 2, July 27, 2002

8/13 of grafts lost to “irreversible rejections” tested positive, compared to 1/7 grafts lost to death with a functioning graftPretransplant titers were much lower (1:50) compared to postrejection titers (>1:2000)


First clinical Studies of MICA Ab testing in renal transplantation IV


In 5 cases of irreversible graft loss the anti-endothelial AB was directed against MICA.


First clinical Studies of MICA Ab testing in renal transplantation V


Functional significance of anti-MICA antibodies: 1. Complement cascade2. Induction of a prothrombotic phenotype in kidney endothelial cells (reduction of antithrombotic and induction of prothrombotic surface molecules)


Detection of anti-MICA relative to the time of transplant I

Time of detection relative to transplantation important to differentiate between auto/heterologous sensitization (antibody present before transplant), and allosensitization (likely when Ab detected after surgery).

Autologous antibodies present before transplant are not DSAs technically (although they may bind to donor antigens).

The clinical scenario of the patient with non-MICA autoantibodies detected before transplant is more likely to be similar to anti-GBM disease post transplant than HLA allosensitization.

On the other hand MICA antibodies detected after surgery could either be bona fide pathogenetic DSAs or immune markers of tissue injury.


Detection of anti-MICA relative to the time of transplant II

Question has been addressed in various studies to data. In (1), sera from patients on the waiting lists, pts with functioning grafts and allograft nephrectomies were examined retrospectively:


In 1 case assessed there was indirect evidence that anti-MICA was a DSA


Detection of anti-MICA relative to the time of transplant III

Anti-MICA antibodies are also detected after transplant.

In a prospective study of 1319 patients assessed for anti-HLA and anti-MICA antibodies anti-MICA was detected in ~9% and was constant over time

(1) American Journal of Transplantation 2007; 7: 408–415


Detection of anti-MICA relative to the time of rejection I

The temporal evolution of anti-MICA positivity was examined retro-spectively in patients with negative pre-txp anti-HLA and graft survival> 1000 days

39 pts who rejected were matched against 26 simultaneous control patients with well functioning grafts.

American Journal of Transplantation 2005; 5: 2265–2272


Detection of anti-MICA relative to the time of rejection II

Antibody could increase or decrease with time and could even disappear.

HLA (+) : 72 % rejection versus 46% (controls)

HLA/MICA (+) : 77% (rejection) versus 46% (controls)

American Journal of Transplantation 2005; 5: 2265–2272

MICA noninc MICA incRejected 8 3Did-not reject 14 0

P-value = 0.005

MICA (-) MICA (+)Rejected 2 9Did-not reject 11 3

P-value = 0.07


Detection of anti-MICA relative to the time of rejection III

Single center retrospective assessment of anti-MICA(+) in 173 patients who received a K or PK (both first transplants and re-transplants )from 1/98-3/03.

Patients classified according to functional status and timing of serum collection :

“Functioning” (n=82) : stable allograft function

“Before – failure” (n=62) : dead or patients with ≥1 rejections

“After – failure” (n=28) : specimen available after patient had returned to dialysis

Biopsies and C4D staining were not uniformly available; authors assumed that all grafts were lost to “chronic rejection”

ISP was the standard protocol of CSA + MMF/AZA + Steroids + Basiliximab induction

Human Immunology 67, 683–691 (2006)


Detection of anti-MICA relative to the time of rejection IV


Anti-HLA was detected with FlowPRA or Luminex.All other Abs were detected by cell dependent cytotoxicity


Summary of early studies regarding the detection of anti-MICA

MICA antibodies may develop before or after transplantation at a low rate (5-20%) and titers may increase after rejection.

Poor graft survival may be associated with positive MICA screens

MICA antibodies do not demonstrate a consistent dose response relationship between graft survival/function and antibody level in either individual patients or at the population level.

On the other hand, there appears to be a dose response curve in anti-MICA titers in anti-HLA(+) patients who lose their grafts.

There is only one ex vivo study looking at complement fixation by anti-MICA isolated from patients (N=5).

Complement fixation by anti-MICA has been shown in heterologous systems(1). In these studies mice were immunized with human MICA antigens followed by assessment of cellular proliferation, cytotoxicity and complement activation. All these functions were enhanced in a MHC-I restricted manner. Note that the MICA locus is absent from the murine genome and the orthologous gene H-2 is 33% identical to human MICA. Is this experiment relevant to clinical scenarios?(1) Human Immunology 67, 215–222 (2006)


Clinical Significance of positive MICA antibody screens pre – transplant I

Retrospective study of 1910 diseased donor kidney transplant recipients recipients who underwent transplantation between 1990 in 20 centers in 13 countries.

Anti-HLA and anti-MICA were determined by ELISA and Luminex assays respectively

Allograft function was assessed by serum creatinine levels at 3,6,12 months. Biopsy findings were not reported

23 pts (1.2%) and 34 (1.8%) were lost to f/u respectively

Patients with anti-MICA antibodies had a 1-year graft-survival rate of 88.3±2.2% as compared with 93.0±0.6% among patients in the MICA antibody–negative group (P = 0.01).

When only recipients of first transplants were considered, the graft survival rate was 87.8±2.4% among 183 MICA antibody–positive patients as compared with 93.5±0.6% among the 1473 recipients who were MICA antibody–negative (P = 0.005).

N Engl J Med 2007;357:1293-300.


Clinical Significance of positive MICA antibody screens pre – transplant II

N Engl J Med 2007;357:1293-300.


Clinical Significance of positive MICA antibody screens pre – transplant III

In summary MICA (+) before transplant was associated with poor graft function.The risk appeared to be higher in patients well matched for the HLA

N Engl J Med 2007;357:1293-300.


Functional Significance of anti-MICA(+) detected after transplantation I

Similar to the case of anti-MICA(+) detected in the pretransplant period, anti-MICA(+) after transplantation is associated with poor outcomes.

Most of the data come from two prospective multicenter study of 1329 and 2389 patients with functioning allografts enrolled in the 13th and 14th International Histocompatibility Workshop (1) . Selection criteria: (-) anti-HLA preTxp and normal renal function 6 months post TxP.

The data from this cohort were analyzing under the assumption that anti-MICA positivity has the same significance as a positive anti-HLA test.

In particular, renal survival of patients with (-) anti-MICA was not compared against patients with (+) anti-MICA but against renal survival of pts with either anti-HLA or anti-MICA positivity.



Functional Significance of anti-MICA(+) detected after transplantation II


Effects of MICA (+) limited to DD recipients


Functional Significance of anti-MICA(+) detected after transplantation III


Effects of MICA (+) limited to DD recipients


Functional Significance of anti-MICA(+) detected after transplantation IV


The only reasonable conclusions that can be drawn from this study are : anti-HLA (+) is associated with poor allograft survival, and that anti-MICA (-) is associated with better survival that anti-HLA (+)


Functional Significance of anti-MICA(+) detected after transplantation V

185 recipients of LR allograft with negative pre-transplant cross match and survival at 6 months post TxP

ISP: Combination of 3 drug regimens, no induction. Rejections Tx with SM pulse x 3 days, and refractory cases with ATG or OCT3

Bx was performed in all patients with unexplained rising Scr.

Banff grade of rejection: I(6), 19(IIA), 16(IIB), 1 (III)



Functional Significance of anti-MICA(+) detected after transplantation V


Overall 16% were anti-MICA (+).40% of anti-MICA(+) patients lost their graft compared to 14% of anti-MICA (-) patients


Relationship between positivity for anti-HLA Abs and anti-MICA I

In (1) looking at pre-transplant MICA sensitivity:

1.9% patients were (+) anti-class I and anti-MICA, and 1.8% harbored both (+) anti-class II and anti-MICA

16.8% (37/220) of patients with anti-class I were MICA(+), versus 10.7% (180/1684) with no anti-class I (p=0.007)

17.2% (35/204) of patients with anti-class II were MICA(+) versus 10.8% (182/1692) of those without anti-class II (p=0.007)

Other studies also showed (2) such an association when anti-MICA specifities were analyzed while others did not examine the issue (3)

In the study (3) with the longest follow-up (10 year), OR(anti-MICA (+)|anti-HLA(+)) was 0.17 (p-value 0.04) in patients who rejected, but 1.79 (p-value=0.66) in patients with functional allografts.

In another study (4) of predominantly anti-HLA (+) (93% out of 266) patients, anti-MICA was detected in 21% with graft loss and 7% without graft loss. All pts with anti-MICA were (+) for anti-HLA

(1) N Engl J Med 2007;357:1293-300. (3) American Journal of Transplantation 2005; 5: 2265–2272(2) Human Immunology 68, 362–367 (2007) (4) Clin Transpl. 2006:265-90


Relationship between positivity for anti-HLA Abs and anti-MICA II


There seemed to an interaction between anti-MICA positivity and anti-HLA positivity in that the % of rejection and graft loss was higher when patients were doubly positive.


Mechanisms responsible for anti-MICA antibody generation I

The following explanations for the development of anti-MICA have been entertained in the literature:

Allosensitization from transfusions (similar to anti-HLA)

Allosensitization from pregnancies

Allosensitization from Donor Antigens (this is the main hypothesis entertained in the transplant literature)

Development as a marker of immune-mediated cell death (e.g. by anti-HLA)

Development as a regulatory mechanism for the innate arm of immunity (NK Tγδ and CD8+ Tαβ cells).

Development as a result of auto-immunity.


Mechanisms responsible for anti-MICA antibody generation I

Anti-MICA as a result of blood transfusion is the least likely explanation (the antigen is not present RBCs, is not expressed at the protein level in lymphocytes or monocytes (or so we think).

Explicitly tested in the pre-transplant series published in N Engl J Med 2007;357:1293-300.

Transfusions were associated with anti-HLA but not anti-MICA antibody formation


Mechanisms responsible for anti-MICA antibody generation II

Prior pregnancies (and multiparity) are plausible explanations for the development of this antibody before transplant in women.

In the pre-transplant series published in N Engl J Med 2007;357:1293-300.there was a slightly higher incidence of anti-MICA positivity in men rather than women

The level of anti-MICA in pregnancy and anti-HLA (-) patients was prospectively examined . Only one specificity could be deemed as significantly different from the controls:



The case of Mr RC (cont'd)

Serum Protein Electrophoresis : decreased globulins, no monoclonal spike

Serum free kappa light chain: 134 (normal < 19.4)

Serum free lambda light chain: 11.4 (normal < 26.3)

UPEP : Possible low concentration of monoclonal protein (but negative immunofixation), and glomerular proteinuria

Urine b2-microglobulin: 2.66 (normal < 0.23)

Serum b2 microglobulin: 10.1 (normal < 2.5)

Severely depressed serum immunoglobulin levels


The case of Mr RC (cont'd)

Bone Marrow Biopsy (9/2/08):

INVOLVED BY A KAPPA LIGHT CHAIN RESTRICTED PLASMA CELL NEOPLASM.

NORMOCELLULAR BONE MARROW WITH TRILINEAGE HEMATOPOIESIS.

MARKEDLY REDUCED IRON STORES.

11.3% Plasma Cells

Patient's final diagnosis was recurrent renal disease due to PCD (multiple myeloma). He was started on Dex/Velcade by Hematology, and has been on Hemodialysis since 10/10/08

Is the MICA antibody causally related to the patient’s graft failure or a coincidence ? Can anti-MICA develop in non transplant settings, and if so what's is function ?


Anti-MICA formation in tumor immuno-surveillance and immunity I

In vitro anti-MICA coating of tumor cells (1) enhances DC tumor antigen uptake and presentation (“DC-vaccine adjuvant”)

Tumour specific CD8 cells are generated at increased frequency after DC priming with anti-MICA

These CD8 cells demonstrated increased tumor cytolytic activity, IFN-gamma production

Tumour specific CD4 responses were also observed

(1) PNAS May 3, 2005 vol. 102 no. 18 6461–6466(2) PNAS June 13, 2006 vol. 103 no. 24 9190–9195


Anti-MICA formation in tumor immuno-surveillance and immunity II

In (2) a patient participating in an experimental protocol for metastatic melanoma with CTL4-Ig (costimulation blockade) was shown to improve after the development of antibodies against the MICA antigen.

Clinical improvement was associated with decreases in soluble MICA antigens and increases in anti-MICA antibody.

Surface expression of NKG2D receptor increased after CLTA4-Ig administration



Anti-MICA formation in tumor immuno-surveillance and immunity III


NK activity was restored to normal in the presence of anti-MICA

IFN-gamma production by CD8(+) T cells against tumor antigens was restored

In summary anti-MICA production by costimulation blockade enhances NKG2D mediated immunity by decreasing the levels of the (immuno-suppressive) tumour derived soluble MICA Ag.


The role of sMICA/anti-MICA in PCD I

(1) PNAS 2008 Jan 29;105(4):1285-90

The role of sMICA/anti-MICA in the progression of PCDs (MGUS to myeloma) was recently examined (1)

Normal plasma cells do not express MICA, where as MGUS plasma cells express surface MICA and myeloma cells expressed cytoplasmic MICA

Soluble MICA was not detected in pts with MGUS but was detected in patients with myeloma.Soluble MICA antigen positivity was associated with depressed NKG2D dependent immune responses


The role of sMICA/anti-MICA in PCD II

(1) PNAS 2008 Jan 29;105(4):1285-90

Opsonization of MICA (+) cells


Anti-MICAs and immunologically mediated native kidney disease I

Patients with Wegener's granulomatosis and other forms of vasculitis demonstrate high numbers of MICA(+) circulating endothelial cells during disease activity (n=16) but not disease remission (n= 36)

These cells produced higher levels of IL-8, ENA-1, MIP-1, and GRO- in active disease compared to remission and healthy controls.

J Am Soc Nephrol 16: 3110-3120, 2005

The numbers of VAP-1/MICA cells were inversely correlated with hemoglobin levels (ρ=0.51, P<0.001) and positively with leukocyte count (ρ=0.5, P = 0.015) and CRP (0.41, P=0.01). Furthermore, VAP-1/MICA cells were significantly and positively correlated with thenumber of organs involved (ρ=0.53, P <0.001)


Anti-MICAs and immunologically mediated native kidney disease II

During active disease, MICA is upregulated in peritubular endothelium and glomerular capillary cells

Patients with active Wegener's will also have a high % of circulating anti-endothelial antibodies (of unknown molecular specificity)

To date the % anti-MICA positivity of patients with Wegener's and vasculitis has not been determined

J Am Soc Nephrol 16: 3110-3120, 2005J Am Soc Nephrol.18(9):2497-508,2007


Conclusions I

MICA antibody positivity is a novel marker associated with graft loss after renal transplant in ~20% of renal transplant patients

Anti-MICA (+) confers poor prognosis irrespective of the time they are detected (before or after transplant)

The source of Anti-MICA(+) pre-Txp remains elusive but interesting possibilities include:

Innate immune system effector functions relating to immunosurveillance of solid tumors and plasma cell disorders

Pathogenetic (auto-antibodies) against renal tissue (as in Wegener's)

A marker of immune cell activation against cell death


Conclusions II

The source of Anti-MICA(+) appearing de-novo post-Txp also remains elusive. It could represent :

A bona fide DSA that requires special attention to immune monitoring after transplant (routine Luminex assays)

A marker of immune cell activation e.g. due to anti- HLA mediated allograft damage

Management of the anti-MICA positive patient in either the pre-op or the post-op setting is far from clear.

Further studies will be needed to examine the relation between, type of native renal disease, occult malignancy and appearance of anti-MICA before transplant.

In the post-op setting, it is not clear whether intensification of ISP is required and which adjunctive therapies should be used. If anti-MICAs are causally implicated in allograft loss , then recombinant sMICA could be a specific therapy

Given preliminary data from native kidney disease, allograft Bx staining for MICA should be examined in a prospective fashion.

mica antibodies in renal transplantation

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